Phospholipase D is an ubiquitous enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid and choline. Its cellular actions are related to the production of phosphatidic acid and include alterations to cell growth, shape, and secretion. There are two mammalian phospholipase D genes whose products (PLD1 and PLD2) are alternatively spliced. Both forms have two highly conserved HKD motifs that are essential for catalysis and dimerization. PLD1 is regulated in vitro and in vivo by protein kinase C and small GTPases of the Rho and ARF families, whereas PLD2 shows a higher basal activity with little or no response to these proteins. The cellular locations and specific functions of the two PLD isoforms remain to be established.